The primary pharmacological target of acetaminophen is prostaglandin endoperoxide H₂ synthase (PGHS). The enzymatic catalytic mechanism is radical-based, initiated, and maintained by the persistent presence of peroxides, particularly peroxynitrite, which is termed "peroxide tone". Whereas the prevailing concept assumes a direct reduction of the active, oxidized enzyme by acetaminophen, here we show that acetaminophen is a potent scavenger of peroxynitrite (peroxynitrite-mediated phenol nitration, IC₅₀72µM; Sin-1-mediated DHR123 oxidation, IC₅₀11µM) and thus inhibits PGHS by eliminating the peroxide tone. Nanomolar concentrations of peroxynitrite increased the activity of isolated PGHS and prostacyclin formation by aortic endothelial cells. This elevated activity was efficiently inhibited by pharmacologically relevant concentrations of acetaminophen (IC₅₀10µM for 6-keto-PGF₁) and other free radical scavengers. However, when the peroxide tone was provided by H₂O₂ or tert-butyl-OOH, acetaminophen had only negligible inhibitory effects. Our concept could help to explain the efficacy of acetaminophen to inhibit PGHS in cell types with moderate oxidant formation. However, high levels of peroxynitrite or other peroxides such as lipid peroxides formed at inflammatory sites might overwhelm the ability of acetaminophen to decrease PGHS activation. The concept presented herein provides a molecular basis to explain the excellent analgesic and antipyretic properties of acetaminophen together with its minimal anti-inflammatory effects.—Schildknecht, S., Daiber, A., Ghisla, S., Cohen, R. A., Bachschmid, M. M. Acetaminophen inhibits prostanoid synthesis by scavenging the PGHS-activator peroxynitrite.